左心室舒張功能不全之分子及臨床研究

Molecular Mechanisms and Clinical Studies of Left Ventricular Diastolic Dysfunction

吳卓鍇(Cho-Kai Wu)

指導教授 ： 江福田 ； 蔡佳醍

國立臺灣大學/醫學院/臨床醫學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.02615

摘要

本研究的目的，主要在於發炎反應物質(proinflammatory cytokines）和左心室舒張功能不良(left ventricular diastolic dysfunction)發生的可能分子機轉。首先研究血漿中的蛋白質對於舒張性心衰竭發生機制的影響，過去的研究顯示SERCA2(sarcoplasmic reticulum Ca2+ ATPase)是影響舒張性心衰竭生成最重要的鈣離子通道，過去的研究也顯示SERCA2的功能跟質量都明顯的影響舒張性心衰竭的發生率。我們的初步研究顯示，血漿中的angiotensin II對於SERCA2促進子(promoter)的影響程度不及tissue necrosis factor (TNF-α)或者interleukin-6 (IL-6)等前發炎反應物質，因此以心肌細胞模式研究TNF-α或者IL-6影響SERCA2表現的訊息傳導模式。我們使用HL-1心肌細胞，複製促進子前1754苷基片段，藉由質體導入HL-1心肌細胞中，接著分別研究TNF-α或者IL-6對於促進子的影響，直接刺激HL-1細胞對於mRNA，蛋白質總量的改變。結果發現TNF-α或者IL-6可以抑制SERCA2促進子的活性乃至於mRNA，蛋白質的量，甚至改變鈣離子通道回收鈣離子的能力(鈣離子電流坡度變得平緩)。接著研究影響SERCA2最強的激素- TNF-α可能抑制SERCA2的機制，同樣使用HL-1細胞，加入TNF-α後研究下游可能的機轉，結果首次發現，TNF-α藉由Nuclear Factor-KappaB (NF-κB) 反應單元結合蛋白機轉抑制SERCA2基因的表現，同時進一步抑制左心室的舒張功能。我們同時也在動物模式中驗證上述細胞模式所發現的機轉，以lipopolysaccharide(LPS)刺激大鼠，引發血漿中的TNF-α上升後上升後，同樣會經由上述的機轉使得大鼠的左心室舒張功能變差，但使用NF-κB抑制劑-PDTC或者simvastatin來抑制發炎反應卻可以在細胞模式中使得此機轉阻斷，SERCA2的量回升。同樣在動物模式下，利用口服的simvastatin給予大鼠服用，抑制血漿中的TNF-α上升，對比對照組的老鼠發現，經由抑制NF-κB反應單元結合蛋白機轉不僅使得SERCA2的量回升，也同樣可以改善左心室的舒張功能不良。
臨床的研究上，也研究血漿中的前發炎反應物質如TNF-α或者IL-6上升和左心室舒張功能各項指標的相關性。首先，我們研究血漿中的TNF-α或者IL-6和心臟超音波下各項標準的評估左心室舒張功能的指標。第一個研究的群體是單純舒張性心衰竭的病患，第二個群組則引入30位加護病房的病患同時具有左心室舒張功能不全者。我們發現在單純舒張性心衰竭病患血漿中的TNF-α或者IL-6和超音波下的左心室舒張功能指標有顯著的相關性(各項指標P<0.01)，同時在嚴重的病患血漿中的發炎反應指數上升，左心室舒張功能也明顯地變差，當病患狀況改善時，發炎指數和左心室舒張功能也平行的發生改善。接著引入了極端發炎反應的病患也就是56位嚴重燒傷的病患，在這樣的族群當中，發現不僅發炎指數更顯著地和左心室舒張功能的超音波指標明顯的相關，同時經過迴歸分析校正之後，左心室舒張功能在這樣的病患更是影響預後的一項獨立因子(HR = 3.99, p = 0.038)。
以上為住院病患具有高發炎反應的患者，為了評估一般族群中前發炎反應是否與左心室舒張功能不全的發生有關，我們選擇臨床門診比較常見，血漿中可能前發炎反應會較正常人高的族群，第一個族群選取了102位肥胖的族群，血漿中的發炎指數顯著的上升，同時以斷層掃描定義內臟脂肪(visceral fat)的多寡，以心臟超音波評估病患是否有左心室舒張功能不全。使用結構方程分析後發現，內臟脂肪主要可以透過發炎反應的產生來導致左心室舒張功能不全的發生，而不是因為脂肪滲透引起心臟功能不全的發生。另外一個族群則選取腹膜透析的病患，我們比較發炎反應，腹膜透析，左心室舒張功能不全三者之間的關係，發現發炎反應本身是和左心室舒張功能不全發生一項獨立的因子，腹膜透析也是和左心室舒張功能不良發生一項顯著的相關因素，同時兩者有交互作用存在，此交互作用可以額外的增加舒張性心臟功能不良發生的機會。最後，我們也研究新的血漿中和組織纖維化相關的激素conntective tissue growth factor (CTGF)，我們利用臨床上台灣舒張性心衰竭群體資料，左心室肥厚的狗模式，心肌細胞模式三種不同的模式來分析，第一次發現血漿中的CTGF和嚴重舒張性心衰竭的心臟超音波E/E’有明顯的相關性存在，另外以核磁共振(MRI)評估左心室的舒張功能也發現，心臟纖維化的程度也和血漿中的CTGF明顯的相關，在動物模式上，組織的CTGF和心臟纖維化的程度經過兩週後平行的上升了，同時CTGF的濃度和血壓的變動有顯著的相關性存在，更是回歸分析後影響心臟舒張功能不良發生的獨立因素。
總結而言，本研究利用動物、細胞模式及基礎分生研究來證實前發炎反應物質(TNF-α)和舒張性心衰竭發生的可能分子機轉，進而發現simvastatin可以阻斷TNF-α訊息傳遞，有治療舒張性心衰竭的效果，再不同程度的發炎反應病患身上驗證了 TNF-α等前發炎反應物質和左心室舒張功能指標的相關程度，並推論出內臟脂肪，腹膜透析都可以透過發炎反應或者交互作用引起左心室的舒張功能不全發生，另外也驗證了新的纖維化指標CTGF可以作為預估左心室舒張功能不全，或者新的治療方法的依據。

關鍵字

左心室舒張功能 ； 發炎反應 ； 心衰竭 ； 分子機轉 ； 臨床研究

並列摘要

The aim of the present doctoral thesis is to investigate the molecular mechanism by which proinflammatory cytokines are involved in the pathegenesis of diastolic heart failure (DHF). We try to find the mechanism by which inflammatory cytokines influence cardiac diastolic function. We realized that the sarcoplasmic reticulum Ca2+ ATPase (SERCA) is essential to the regulation of intracellular calcium levels in cardiac and is crutial for the development of DHF. Our pilot study reveal that Angiotensin II does not appear to exert an effect on SERCA2 levels in cardiomyocytes, but pro-inflammatory cytokines such as tissue necrosis factor-alpha (TNF-α) or IL-6 have even greater effect over SERCA2 expression in cardiomyocyte models. Regarding TNF-α or IL-6 and SERCA2 signal transduction, we investigated whether TNF-α or IL-6 modulates SERCA2 calcium current through transcriptional regulation, using a HL-1 cells model. We cloned a 1754 bp promoter fragment of rat SERCA2 gene and amplified by polymerase chain reaction (PCR). We determined that TNF-α or IL-6 decreased SERCA2 mRNA, protein levels and SERCA2 promoter activities, which resulted in an attenuation calcium transient. Accordingly, in the following study, we explored the pathway of TNF-α on the transcriptional regulation of SERCA2 cardiomyocytes. We first found that TNF-α decreased SERCA2 gene expression and induced left ventricular (LV) diastolic dysfunction through Nuclear Factor-KappaB (NF-κB) Element–Binding Protein–Dependent Pathway. We also used an in vivo rat model of hyper-TNF-α to verify the results obtained in the cellular study. The upstream NF-κB dependent pathway was critical for TNF-α–induced decreased transcription of SERCA2 gene. The NF-κB blocker (PDTC) and pre-incubation of simvastatin inhibited TNF-α–induced NF-κB dependent pathway and augmented expression of SERCA2 gene which in turns lead to improvement of LV diastolic function in a in-vivo rat model of LPS injection.
Regarding clinical studies, to evaluate the association between cytokines (TNF-αor IL-6) and left ventricular dysfunction indices, first, we sought to assess plasma levels of IL-6 and TNF-α in patients with DHF, including critically ill patients. We included several goups of patients. The first group consisted of pure DHF patients, and the second group enrolled 30 consecutive patients with left ventricular diastolic dysfuction that were admitted to the intensive care units (ICUs). Significant correlations (p < .01 for each) were found for TNF-α, IL-6 and cardiac diastolic dysfunction indices. Cytokine levels were also correlated with diastolic function in critical ill patients, and diastolic function improved significantly in association with decrease of cytokines. We later included 56 critically burned patients and we found that there was a significant correlation between LV diastolic dysfunction and in-hospital mortality in critically burned patients (HR = 3.99, p = 0.038) after risk factors adjusted. Regarding the patients in the general population with higher inflammatory status, we further included 2 other goups of patients to prove the above hypothesis. First we we included 102 obese adults. In this study, we found a significant association between LV diastolic dysfunction and fat content as measured by CT. With the analysis, we verified that fat deposition acts mainly through inflammation to affect LV diastolic dysfunction and less by its own effect (e.g. fat infiltration of LV). We also tried to establish the association between inflammation and LV diastolic dysfunction in peritoneal dialysis (PD) and non-PD patients. We established that an interaction between PD and inflammation, especially TNF-α, was also shown to further aggravate LV diastolic dysfunction. Finally, we investigated an emerging marker for tissue fibrosis, connective tissue growth factor (CTGF) and its association with cardiac diastolic function using cellular and animal models and clinical human data. Significant correlation was found between plasma CTGF and E/e’in DHF patients. Severity of cardiac fibrosis evaluated by CMRI also correlated with CTGF. In the cell model, stretch increased secretion of CTGF from cardiomyocytes. In the canine model, myocardial tissue CTGF expression and fibrosis significantly increased after 2 weeks of aortic banding.
In conclusions, the present doctoral thesis combined animal, cell models and molecular studies to demonstrate how cytokines (including TNF-α, IL-6 and CTGF) are involved in the pathogeneses of cardiac fibrosis, SERCA2 regulation, which are important substrates of DHF. Current study also discovered that simvastatin could block TNF-αsignal transduction which could be beneficial for the treatment of DHF. By testing the association of TNF-α and LV diastolic dysfunction parameters in patients with different severities of DHF, we could conclude that both visceral fat, PD could interact with inflammation, which in turns, lead to the development of LV diastolic dysfunction. Finally, we provided evidence that CTGF could be a marker for LV diastolic dysfunction and also targets of treatment.

並列關鍵字

Left ventricular diastolic function ； inflammation ； heart failure ； molecular mechanism ； clinical studies

參考文獻

(1998). "How to diagnose diastolic heart failure. European Study Group on Diastolic Heart Failure." Eur Heart J 19(7): 990-1003.

Abe, T., Y. Ohga, et al. (2002). "Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats." Am J Physiol Heart Circ Physiol 282(1): H138-148.

Ammar, K. A., M. M. Redfield, et al. (2008). "Central obesity: association with left ventricular dysfunction and mortality in the community." Am Heart J 156(5): 975-981.

Angeja, B. G. and W. Grossman (2003). "Evaluation and management of diastolic heart failure." Circulation 107(5): 659-663.

Asahi, M., H. Nakayama, et al. (2003). "Regulation of sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase by phospholamban and sarcolipin: implication for cardiac hypertrophy and failure." Trends Cardiovasc Med 13(4): 152-157.